Figure 4.

Chemically stabilized FM-miR-34a has increased stability and activity

(A) miR-34a was modified on all ribose sugars at the 2′ position and select phosphodiester bonds were changed to more stable phosphorothioate bonds to generate a FM-miR-34a. Modification positions are indicated in the schematic. (B) Both miR-34a and FM-miR-34a were incubated in the presence of 50% FBS for the indicated times followed by resolving on glycerol tolerant polyacrylamide gels. Shown is a representative image from at least three independent biological replicates. The top band, which represents the intact miR-34a is quantified in the graph. (C and D) LNCaP (C) or 22RV1 (D) cells were transfected with the indicated concentrations of miR-34a or FM-miR-34a or 50 nM of NC or miR-34a mimic (Mimic). The indicated target genes were evaluated by immunoblot 72 h later. Values below each lane represent band intensity normalized to β-actin and relative to UT (untreated). (E) Representative images (left) and quantification (right) of LNCaP cells that invaded through a matrix over 96 h following transfection with miR-34a or FM-miR-34a for 48 h prior to seeding in transwell chambers (n = 2). ∗ p < 0.05, one-way ANOVA. (F) Targeted silencing of miR-34a Renilla sensor after the transfection of LNCaP miR-34a-sensor-cells with 50 nM DUPA-FM-miR-34a, DUPA-nigericin-FM-miR-34a, or respective controls for 48 h. Data represent two biological replicates with at least six technical replicates each. ∗∗∗∗p < 0.0001, one-way ANOVA. Error bars are mean ± SD, significance was determined relative to untreated. nig, nigericin. (G) miR-34a was quantified using qRT-PCR from the cytosolic fractions obtained from LNCaP cells treated with either 100 nM DUPA-FM-miR-34a, DUPA-nigericin-FM-miR-34a, or their respective controls for 18 h. Experiment repeated twice with three technical replicates in each experiment. ∗∗p < 0.01, one-way ANOVA. Error bars are mean ± SD; nig, nigericin.